1. Field of the Invention
The present invention relates to a method for regulating cell death. More specifically, the present invention relates to a method for providing a plant to which is conferred resistance to various environmental stresses by regulating an expression level of a cell death regulatory gene.
2. Description of the Related Art
When a plant is infected with a pathogen, e.g., virus, bacteria, filamentous fungi, and viroid, the plant shows either of the following reactions: 1) allowing pathogen to grow by spreading through the entire body of the plant, whereby the plant gets disease or 2) enclosing pathogen in an infected site so as to prevent if from spreading through the entire body of the plant, whereby the plant is provided with resistance to the pathogen. The latter reaction of a plant against the pathogen is called a hypersensitive response or reaction (HR). It is known that, in this reaction, cell death locally occurs in an infected site to form necrotic lesions. Such a formation of necrotic lesions involved in pathogen infection is a typical resistance reaction of a plant, which is considered as an example of programmed cell death. However, the molecular mechanism of this reaction remains unclear.
The HR does not occur in all plants. The HR is believed to occur when a plant intrinsically contains a gene which recognizes a product of a pathogenic gene derived from infecting pathogen. In the case where such a gene is not present, the HR does not occur, and a plant is not resistant against the pathogen infection.
The HR of tobacco against tobacco mosaic virus (TMV) infection is a model system which has been conventionally used for studying the HR of a plant.
An N gene is one of the cell death regulatory genes involved in the HR (i.e., cell death) due to TMV infection. It is reported that tobacco having the N gene (NN tobacco) shows the HR against TMV infection, but tobacco having no N gene (nn tobacco) does not show the HR (Holmes, Phytopathology, 28, 553, (1938)). The HR of the NN tobacco occurs only at 24xc2x0 C. or lower. It does not occur at 28xc2x0 C. or higher. Therefore, it has been considered that both the N gene and the temperature condition are required for inducing the HR in a TMV-infected cell.
However, the inventors"" group has found that, in the case where the NN tobacco is treated with actinomycin D (AMD) and heat (50xc2x0 C., 2 minutes), the HR is induced in the NN tobacco against TMV infection even under the temperature condition of 30xc2x0 C. at which the HR does not usually occur. Furthermore, the HR was also induced against TMV infection in the nn tobacco having no N gene, in the case where the nn tobacco was similarly treated with AMD and heat. Because of this, it was clarified that cell death against TMV infection may occur irrespective of the presence or absence of the N gene and the temperature condition (Shimomura and Ohashi, Virology, 43, 531, (1971); Ohashi and Shimomura, Virology, 48, 601 (1972)). It is known that AMD inhibits DNA-dependent RNA synthesis in a nucleus (Reich et al., Proceedings of the National Academy of Sciences, 48, 1238 (1962)). Thus, a possibility was shown that a novel cell regulatory gene may be present in a plant, and that the HR may be induced by suppression of transcription of the gene, followed by suppression of synthesis of proteins.
It is considered that if the above-mentioned cell death regulatory gene is identified, cell death of a plant can be regulated (promoted or suppressed) by controlling an expression level of the gene. In particular, it is an important task in the agricultural field to provide a plant which is conferred with resistance to environmental stress by regulating cell death.
However, the cell death regulatory gene as described above has not been identified. To the extent that the inventors are aware, there has been no study for providing a plant which is conferred environmental-stress resistance by regulating an expression level of such a gene to promote or suppress cell death.
The present invention provides a method for regulating cell death in a plant of the present invention including the steps of: transforming a plant cell with a polynucleotide containing a gene encoding DS9 or a homologue thereof or a part of the gene; and redifferentiating the transformed plant cell to obtain a plant, wherein the DS9 or the homologue thereof is an ATP-dependent Zn-type metalloprotease, and the polynucleotide decreases or increases production of the ATP-dependent Zn-type metalloprotease in the plant cell, whereby cell death of a cell in the plant is promoted or suppressed.
A polynucleotide containing a gene encoding DS9 or a homologue thereof or a part thereof may be incorporated into a DNA in a nucleus of a plant cell by a known gene recombinant technique. The term xe2x80x9cpolynucleotidexe2x80x9d refers to a polymer of nucleotides, and is not limited to a particular chain length.
In one embodiment of the present invention, the polynucleotide contains the gene encoding the DS9 or the homologue thereof or the part of the gene in an antisense orientation, whereby cell death of a cell in the plant is promoted.
A method for producing a plant which is conferred with resistance to environmental stress of the present invention includes the steps of: transforming a plant cell with a polynucleotide containing a gene encoding DS9 or a homologue thereof or a part of the gene; and redifferentiating the transformed plant cell to obtain a plant, wherein the DS9 or the homologue thereof is an ATP-dependent Zn-type metalloprotease, and the polynucleotide decreases or increases production of the ATP-dependent Zn-type metalloprotease in the plant cell.
In one emboidment of the present invention, the environmental stress is pathogen infection.
In another embodiment of the present invention, the polynucleotide contains the gene encoding the DS9 or the homologue thereof or the part of the gene in an antisense orientation.
In another embodiment of the present invention, the homologue has a homology of about 70% or more with respect to an ATPase region of the DS9.
In a method for screening a selective inhibitor of a gene encoding DS9 or a homologue thereof of the present invention, the DS9 or the homologue thereof is an ATP-dependent Zn-type metalloprotease, wherein the method includes the steps of: introducing a candidate inhibitor into an expression system having a gene encoding the DS9 or the homologue thereof; and identifying whether or not production of the DS9 or the homologue thereof is selectively decreased in the expression system.
Thus, the invention described herein makes possible the advantages of (1) providing a method for promoting or suppressing cell death by regulating an expression level of a cell death regulatory gene; and (2) providing a method for producing a plant which is conferred with resistance to environmental stress, e.g., pathogen infection, by regulating cell death; and (3) providing a method for screening a selective inhibitor of a cell death regulatory gene.